Method and compositions for inhibiting thrombin-induced coagulation

ABSTRACT

A method of achieving safe and effective treatment or prevention of potentially harmful blood clots, or in inhibiting the coagulation of blood when so desired such as during a wide array of disease conditions including stroke, myocardial infarction, sickle-cell crisis and venous thrombosis, is provided by the administration of a fibrinogen-binding protein capable of binding at the N-terminal Bβ chain of fibrinogen, such as SdrG or Fbe, or their respective binding regions such as the A domain. In addition, compositions comprising effective amounts of the fibrinogen-binding proteins are also provided. The present anti-coagulation compositions have been shown to inhibit thrombin-induced fibrin clot formation by interfering with the release of fibrinopeptide B and the resulting anti-coagulation effects can be achieved without potential for causing or exacerbating unwanted side effects such as thrombocytopenia associated with prior art anticoagulants such as heparin.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional PatentApplication No. 60/290,072, filed May 11, 2001.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] The subject matter of this application was supported by Grant No.AI20624 from the National Institutes of Health.

FIELD OF THE INVENTION

[0003] The present invention relates in general to SdrG, afibrinogen-binding bacterial adhesin, and in particular to the use ofSdrG or its binding region as an anti-coagulation agent by virtue of itsability to inhibit thrombin-induced fibrin clot formation by interferingwith the release of fibrinopeptide B. In addition, the invention relatesto methods and compositions utilizing SdrG or its binding region fortreating or preventing thrombin-induced coagulation and conditionsassociated therewith.

BACKGROUND OF THE INVENTION

[0004] Coagulase-negative staphylococci (CNS) are importantopportunistic pathogens that are particularly associated with foreignbody infections in humans. Staphylococcus epidermidis is the most commonpathogenic species of CNS and accounts for 74-92% of the infectionscaused by this group of staphylococci (1). The molecular pathogenesis ofmost infections is complex and involves multiple microbial factors andhost components, but is generally initiated by the adherence of themicrobe to host tissues. Bacterial adherence involves specific surfacecomponents called adhesins, and bacterial pathogens, such asstaphylococci that live in the extracellular space of the host, targetextracellular matrix (ECM) components, including fibrinogen (Fg) andfibronectin, for adherence and colonization. This process is mediated bya sub-family of adhesins that have been termed MSCRAMM®s (microbialsurface components recognizing adhesive matrix molecules) (2).Staphylococcus aureus expresses multiple MSCRAMM®s of which several havebeen characterized in some detail (For a recent review see Ref. 3), andvarious MSCRAMM®s have been the subject of U.S. Patents, includingfibronectin binding proteins such as disclosed in U.S. Pat. Nos.5,175,096; 5,320,951; 5,416,021; 5,440,014; 5,571,514; 5,652,217;5,707,702; 5,789,549; 5,840,846; 5,980,908; and 6,086,895; fibrinogenbinding proteins such as disclosed in U.S. Pat. Nos. 6,008,341 and6,177,084; and collagen binding proteins as disclosed in 5,851,794 and6,288,214; all of these patents incorporated herein by reference. Inaddition, other information concerning SdrG and other MSCRAMM®s can befound in U.S. Ser. No. 09/810,428, filed Mar. 19, 2001, incorporatedherein by reference; and U.S. Ser. No. 09/386,962, filed Aug. 31, 1999,incorporated herein by reference.

[0005] In addition to S. epidermidis, S. aureus also causes seriousforeign body infections. S. aureus appears to adhere to the biomaterialthrough an indirect mechanism. Upon implantation, the foreign bodyrapidly becomes coated with host proteins derived primarily from plasmawith Fg being a dominant component. S. aureus appears to adhere to theabsorbed proteins rather than to the biomaterial itself using adhesinsof the MSCRAMM® family (4, 5). At least four of the S. aureus MSCRAMM®srecognize Fg. Two of these MSCRAMM®s, clumping factor A and B (ClfA ,ClfB ), have Fg-binding A-regions followed by a long segment of Ser-Asp(SD) dipeptide repeats. The other two Fg-binding MSCRAMM®s, contain asimilar ligand binding A-region followed by a fibronectin binding motifthat is repeated 5 times (6). Because the fibronectin binding activitywas identified first, these two MSCRAMM®s are known as fibronectinbinding protein A and B (FnbpA and FnbpB) (7, 8). Studies havedemonstrated the importance of ClfA and ClfB in the adherence of S.aureus to plasma-coated biomaterials. S. aureus mutants deficient in oneor both of these MSCRAMM®s exhibited an impaired ability to adhere toplasma-coated catheters in vivo or ex vivo (9, 10).

[0006] For S. epidermidis, adherence to foreign bodies appears toinvolve both specific and non-specific processes. The bacteria mayinitially associate directly with the foreign body through non-specificinteractions, while the later stages of adherence may involve morespecific interactions between bacterial adhesins and host ligands. S.epidermidis expresses polysaccharide adhesins including PS/A and PIA,which are encoded by the ica locus (11, 12). In addition, the presentinventors (13) and others (14) have recently shown that S. epidermidiscontains surface proteins structurally related to S. aureus MSCRAMM®s.Two of these S. epidermidis proteins, called SdrF and SdrG, havefeatures typical of Gram-positive bacterial proteins that are anchoredto the cell wall. Both proteins show significant amino acid sequencehomology to ClfA and ClfB from S. aureus including an ˜500 amino acidlong A region, a SD dipeptide repeat region and features required forcell wall anchoring, including a LPXTG (SEQ ID NO: 1) motif (FIG. 1A).Recent studies by Pei, et al. suggest that another S. epidermidisprotein, called Fbe, can bind Fg and, much like SdrG, specificallyrecognizes the Bβ chain of this molecule (15). However, this referencedoes not disclose or suggest the specific binding site for the Fbeprotein on fibrinogen and thus does not disclose or suggest that thebinding site for this protein would be related to of affect in anymanner the binding site for thrombin on fibrinogen.

[0007] Of these proteins from S. epidermidis, SdrG is of particularinterest for its ability to bind Fg. Fg is known to play a critical rolein the formation of blood clots, although previously the precise bindingsite of SdrG to Fg has not been localized with specificity. Accordingly,because the precise binding site for SdrG in the Fg Bβ chain has notbeen localized, it has not been previously been associated with thethrombin cleavage site on fibrinogen and thus it has not previously beenrecognized or suggested that SdrG might be useful in inhibiting thethrombin-induced cleavage of fibrinogen and the thrombin-induced processof clot formation.

[0008] In general, the blood clots generated by Fg, e.g. through itscleavage by thrombin to form fibrin and start the process of bloodcoagulation, are beneficial in the normal wound healing process.However, abnormal clots caused by the cleavage of Fg can lead tothrombosis, a condition where a clot develops in the circulatory system.Thrombosis is an extremely dangerous condition and may produce ischemicnecrosis of the tissue supplied by the artery, e.g., myocardialinfarction due to thrombosis of a coronary artery, or stroke due tothrombosis of a cerebral artery. In addition to the above, venousthrombosis may cause the tissues drained by the vein to become edematousand inflamed, and thrombosis of a deep vein may result in a pulmonaryembolism. Still other problems result in sickle-cell patients whereinthe malformed “sickle cells” can also lead to a sickle-cell crisis statein which coagulation reaches dangerous proportions, and this can onceagain result in serious injury or even death.

[0009] Generally, anticoagulant agents such as heparin and itsderivatives are used to treat thrombosis and to prevent or reducecoagulation when desirable such as in the case of myocardial infarctionand the other conditions discussed above. Heparin works by inhibitingthrombin generation and in antagonizing thrombin's action. However, theuse of heparin has distinct problems which have yet to be overcome. Onedisadvantage associated with heparin is that it can only be administeredparenterally. Another serious disadvantage is major bleeding occurs in1% to 33% of patients who receive various forms of heparin therapy. Infact, purpura, ecchymoses, hematomas, gastrointestinal hemorrhage,hematuria, and retroperitoneal bleeding are regularly encounteredcomplications of heparin therapy. In addition to the abovecomplications, thrombocytopenia occurs in 1% to 5% of patients receivingheparin.

[0010] Accordingly, there is thus a distinct and growing need to providealternatives to heparin as anti-coagulation agent which do not sufferfrom all of the above-mentioned side effects or disadvantages. One suchalternative is the use of snake venom products including ancrod, anα-fibrinogenase isolated from Calloselasma rhodostoma (Malayan Pitviper). However, ancrod appears to release only FpA and leads to theformation of an unstable fibrin clot (Bell 1997). Moreover, because thisdefibrinating enzyme cleaves FpA and not FpB from Fg, it forms a clotthat is very sensitive to endogenous fibrinolysis, and additionallyactivates plasminogen further contributing to fibrinolysis (Pizzo,Schwartz et al. 1972; Carr 1975; Bell 1997). Hypofibrinogenemia, i.e.,the reduction of Fg in the blood, must be sustained by administeringancrod daily since after termination of treatment, the plasma Fg risesand returns to normal levels in days (Bell, Bolton et al. 1968). Thelimited clinical experience indicates that which defibrination may beachieved with ancrod, the elaboration of neutralizing antibodies withrepeated injections of ancrod leads to resistance (see, e.g., Pitney,Holt et al. 1969; Pitney and Regoeczi 1970), (Vinazzer 1973; Sapru, Mozaet al. 1975) .

[0011] In short, there is a distinct and acute need to provide a safeand effective alternative to the use of heparin in achieving theinhibition in blood coagulation in cases wherein such inhibition may becrucial in saving the life of a human or animal patient.

SUMMARY OF THE INVENTION

[0012] It is thus an object of the present invention to provide safe andeffective alternatives to the use of heparin in achieving therapeuticanti-coagulant treatment in human and animal patients.

[0013] It is further an object of the present invention to providemethods of utilizing fibrinogen-binding proteins from S. epidermidis inthe prevention or treatment of thrombin-induced coagulation in human oranimal patients.

[0014] It is further an object of the present invention to providemethods of utilizing fibrinogen-binding proteins from S. epidermidis inorder to reduce or prevent thrombin-induced coagulation and to enhancethe dissolution of blood clots in human or animal patients.

[0015] It is another object of the invention to provide therapeuticcompositions based on fibrinogen-binding proteins from S. epidermidiswhich bind to the Bβ chain of fibrinogen which are useful in preventingor treating thrombin-induced coagulation in human or animal patients inneed thereof.

[0016] It is still further an object of the present invention to developcompositions from fibrinogen-binding proteins from S. epidermidis whichbind to the Bβ chain of fibrinogen, and which can block the thrombinbinding site on fibrinogen so as to be useful in methods of preventingcleavage of fibrinogen by thrombin and inhibiting the release offibrinopeptide B from fibrinogen.

[0017] These and other objects are provided by virtue of the presentinvention which comprises compositions and methods which utilize theSdrG protein from S. epidermidis, and other proteins which bind to theBβ chain of fibrinogen such as the A region of SdrG and the Fbe protein,in order to treat or prevent thrombin-induced coagulation in human oranimal patients. In addition, the invention comprises methods ofadministering SdrG so as to treat or prevent a wide variety ofconditions wherein blood coagulation can be dangerous or evenlife-threatening coagulation, including venous thrombosis, myocardialinfarction and sickle cell crisis episodes. The invention utilizes theability of SdrG to inhibit thrombin-induced fibrin clot formation byinhibiting thrombin binding to fibrinogen and interfering with therelease of fibrinopeptide B, and therapeutic compositions containing aneffective amount of SdrG can thus be used as effective anti-coagulationagents. The SdrG compounds and compositions of the present invention mayalso be used to reduce the concentration of plasma fibrinogen in apatient's blood when so desired.

[0018] These embodiments and other alternatives and modifications withinthe spirit and scope of the disclosed invention are described in, orwill become readily apparent from, reference to the detailed descriptionof the preferred embodiments provided herein below.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

[0019]FIG. 1 is a representation of the structural organization of SdrGwherein FIG. 1A is a representation of SdrG. The number of amino acidresidues contained in each region is indicated below each segment. S,signal sequence, A, N-terminal Fg binding region, B1 & B2, repeats ofunknown function, R, serine-aspartate repeat region, W, wall-spanningregion, M, membrane-spanning region. The positively-charged tail andLPXTG motif involved in cell-wall anchoring are also indicated; FIG. 1Bis a model of the recombinant His-tag construct rSdrG(50-597),representing the A region; and FIG. 1C shows a Coomasie stained SDS-PAGEof purified rSdrG(50-597).

[0020]FIG. 2 is a graphic representation of tests showing rSdrG(50-597)binding to immobilized Fg. Increasing concentrations of rSdrG(50-597)() were incubated with immobilized Fg in an ELISA. After incubation inthe wells for 1 h at room temperature, bound protein was detected asdescribed in the materials and method section. The apparent K_(D) was0.9×10⁻⁷ M. Values represent the mean±standard deviation of triplicatewells.

[0021]FIG. 3 is a graphic representation of tests showing localizationof the rSdrG(50-597) binding site in Fg using recombinant Bβ chainconstructs in accordance with the invention. In FIG. 3A, models of therecombinant truncates of the Fg Bβ chain constructed using the pQE30His-tag vector or the GST fusion vector PGEX-KG are shown. In FIG. 3B,whole E. coli cell lysates containing the recombinant proteins wereloaded onto a 10% SDS-polyacrylamide gel. The gel was transferred to anitrocellulose membrane and the blot was probed with biotin labeledrSdrG(50-597) and developed as described in the materials and methodssection. Lane 1, native Fg, lane 2, rβ(1-462), lane 3, rβ(1-341), lane4, rβ(1-220), lane 5, rβ(1-195), lane 6, rβ(25-195), lane 7, rβ(1-95),lane 8, rβ(25-95).

[0022]FIG. 4 shows results of tests indicating inhibition ofrSdrG(50-597) binding to immobilized Fg by synthetic peptides inaccordance with the present invention. In these tests, rSdrG(50-597) (50nM) was pre-incubated with increasing concentrations of peptides for 1 hat room temperature and transferred to microtiter wells coated with 1 μghuman Fg. After incubation in the wells for 1 h at room temperature,bound SdrG was detected as described in the materials and methodssection. For FIG. 4A: β1-25 (), β6-25 (Δ), β1-25S (♦); FIG. 4B: β6-20(▪), β1-20 (▾) and β11-20 (◯). For FIG. 4C: FpA (□), FpB (▴), β1-25 (),β1-25S (♦). Values represent the mean±standard deviation of triplicatewells.

[0023]FIG. 5 shows rSdrG(50-597) binding to thrombin digested Fg. Fgcoated microtiter wells were pretreated for 30 min at 37° C. withthrombin (▴), thrombin and hirudin (), hirudin alone (♦) or untreated(▪). Plates were blocked, washed and incubated with biotin labeledrSdrG(50-597) (25-1000 nM) for 1 h at room temperature. Bound SdrG wasdetected as described in materials and methods. Values represent themean±standard deviation of triplicate wells.

[0024]FIG. 6 is a quantitative analysis of rSdrG(50-597) binding tointact immobilized Fg or Fg peptide β1-25. FIG. 6A shows increasingconcentrations of rSdrG(50-597) were incubated with thefluorescein-labeled N-terminal Bβ chain peptide β1-25 (10 nM) for 3 h inthe dark at room temperature. Equation 1 was used to fit the bindingdata. From three experiments the K_(D) for the interaction ofrSdrG(50-597) with peptide β1-25 was calculated to be 1.4±0.01×10⁻⁷ M.FIG. 6B: Binding of the fluorescein-labeled β1-25 to rSdrG(50-597) inthe presence of increasing concentrations of unlabeled β1-25 () or thescrambled Bβ chain peptide β1-25S (▴). Values are the mean of duplicatereactions.

[0025]FIG. 7 shows the inhibition of fibrin clot formation byrSdrG(50-597). Thrombin (1.0 NIH unit/ml) was added to a mixture of Fg(3.0 μM) and rSdrG(50-597) () (0-6.0 μM) or BSA (▴) (0-6.0 μM) inmicrotiter wells. Fibrin clot formation was monitored by measuring anincrease in optical density at 405 nm. Values represent themean±standard deviation of quadruple wells.

[0026]FIG. 8 shows the Inhibition of FpB release by rSdrG(50-597).Superimposed chromatograms show the amount of fibrinopeptide releasedwhen the Fg-thrombin sample has no SdrG present (upper curve) and whenthe Fg-thrombin sample is incubated with 1.5 μM rSdrG(50-597) (lowercurve) at the 60 min time point. The decrease in the amount of FpBreleased with SdrG present is shown in the lower curve.

[0027]FIG. 9 shows rSdrG(50-597) binding to Serine protease-digested Fg.Fg coated microtiter wells were pretreated for 30 min at roomtemperature with ancrod (), PBS (untreated) (▴), thrombin (♦) orcontortrixobin (▪). Plates were blocked, washed and incubated with aserine protease inhibitor (1 NIH unit/ml hirudin for thrombin and 100μg/ml PMSF for ancrod and contortrixobin). Biotin labeled rSdrG(50-597)(25-1000 nM) was incubated in the wells for 1 h at room temperature.Values represent the mean±standard deviation of triplicate wells.

[0028]FIG. 10 shows the inhibition of FpB Release by rSdrG(50-597).Superimposed chromatograms show the amount of fibrinopeptide releasedwhen the Fg-contortrixobin sample has no SdrG present (upper curve) andwhen the Fg-contortrixobin sample is incubated with 1.5 μM rSdrG(50-597)(lower curve) at the 60 min time point. The decrease in the amount ofFpB released with SdrG present is shown in the lower curve.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] In accordance with the present invention, there is provided amethod and composition for treating or preventing thrombin-inducedcoagulation wherein an effective amount of SdrG which can bind the Bβchain of fibrinogen, or its active fragments such as the A region ofSdrG, is administered so as to inhibit the binding of thrombin tofibrinogen and reduce or prevent the release of fibrinopeptide B fromfibrinogen and thus prevent the thrombin-induced clot formation process.In accordance with the present invention, the inventors have nowlocalized the fibrinogen-binding site for SdrG to the Bβ chain of theN-terminal region of Fg, and more particularly to the region of fromabout residues 6-20 on the Fg Bβ chain which is proximal to the thrombincleavage site. Accordingly, the present invention provides for methodsof using SdrG as an anti-coagulation agent since it inhibits thrombinbinding, thus preventing the cleavage and subsequent release offibrinopeptide B which is an initial step in the initiation of theproduction of fibrin and the thrombin-induced formation of blood clots.

[0030] SdrG is a fibrinogen-binding protein from S. epidermidis whichhas a binding region known as the A region or A domain at residues50-597 of the SdrG protein. Detailed information concerning SdrG and itsprimary binding region, known as the A domain or A region, has beendisclosed in pending U.S. Ser. No. 09/386,962, filed Aug. 31, 1999,incorporated herein by reference. The SdrG protein suitable for use inthe present invention, which includes binding regions of the SdrGprotein including the A domain or region, may be prepared throughisolation of the natural protein or binding region, or more preferablythrough recombinant means using nucleic acids coding for SdrG and/or itsbinding region A. The nucleic acid and amino acid sequences for SdrG andits binding region A have been previously disclosed in pending U.S. Ser.No. 09/386,962, filed Aug. 31, 1999 as discussed above, and thesesequences may be utilized in conventional recombinant procedures inorder to produce SdrG and/or its binding region A which will be suitablefor use in the present invention.

[0031] In accordance with one specific embodiment of the presentinvention, a recombinant SdrG A region was obtained using plasmidcloning of an sdrG gene fragment. In this procedure, Escherichia colistrain JM101 was used for plasmid cloning. E. coli strain Topp3(Stratagene) was used for protein expression. Strains harboring plasmidswere grown in Lennox L broth (Sigma) or on Lennox L agar (Sigma)supplemented with 100 μg/ml ampicillin. The gene fragment encoding theentire A-region was amplified by PCR using S. epidermidis K28 genomicDNA as a template. The oligonucleotide primers used were5′-CCCGGATCCGAGGAGMTACA GTACAAGACG-3′ (SEQ ID NO: 2) and5′-CCCGGTACCGATTTTTTCAGGAGGCAAGTCACC-3′ (SEQ ID NO: 3). The restrictionenzyme cleavage sites (underlined) BamHI and KpnI were incorporated intothe forward and reverse primers, respectively. The reactions werecarried out using a Perkin-Elmer DNA thermocyclcer. The reactionscontained 50 ng of template DNA, 100 pmol of forward and reverseprimers, 20 mM Tris-HCl (pH 8.8), 2 mM MgSO₄, 10 mM KCl, 10 mM(NH₄)₂SO₄, 0.1% Triton X-100, 25 mM of each dNTP, and 5 units of Pfu DNApolymerase (Stratagene). Amplification was performed at 94° C. for 1min, 50° C. for 1 min, 72° C. for 4 min, for 25 cycles.

[0032] Next, the cloning of sdrG into the expression plasmid was carriedout. In this process, the amplified sdrG fragment was digested withBamHI and KpnI and ligated into the expression plasmid pQE30 (QiagenInc.) that had been digested with the same enzymes, yielding theconstruct pSdrG(50-597). The recombinant protein rSdrG(50-597) expressedfrom this plasmid contains an N-terminal extension of six histidineresidues (His-tag). Expression and purification of the recombinantprotein was then obtained. In these steps, E. coli transformed withpSdrG(50-597) was grown for ˜2 h for the cultures to give an OD₆₀₀ of0.6. rSdrG(50-597) expression was induced by the addition ofisopropyl-β-thiogalactopyranoside (IPTG) (Gibco-BRL) (225 μM) and thecultures were incubated at 37° C. for an additional 3 h. Bacteria werepelleted and resuspended in phosphate buffered saline (PBS), pH 7.5 (140mM NaCl, 270 μM KCl, 430 μM Na₂HPO₄, 147 μM KH₂PO₄) and frozen o/n at−20° C.

[0033] Next, bacterial cells were thawed and mechanically lysed by usinga French Pressure Cell (SLM Amnico). Cell debris was removed bycentrifugation and filtration through a 0.45 μm filter membrane. Thesupernatant containing the recombinant protein was applied to a Ni²⁺charged (87.5 mM) 5 ml Hi Trap chelating column (Amersham PharmaciaBiotech) connected to a FPLC system. The column was equilibrated withbuffer A (0.1 M NaCl, 10 mM Tris-HCl, pH 8.0) before the application ofthe filtered lysate. The column was then washed with 10 bed volumes ofbuffer A containing 5 mM imidazole. Bound protein was eluted with acontinuous linear gradient of imidazole (5-120 mM; total volume 160 mls)in buffer A. Fractions were monitored for protein by determining theabsorbance at 280 nm and fractions containing rSdrG(50-597) wereidentified by SDS-PAGE (16). These fractions were pooled and dialyzedagainst PBS, pH 7.5. The dialyzed protein was then applied to aQ-Sepharose column (Amersham Pharmacia Biotech) equilibrated with 25 mMTris-HCl, pH 8.0. Bound protein was eluted with a continuous lineargradient of NaCl (0-0.5 mM; total volume 160 mls) in 25 mM Tris-HCl, pH8.0. Fractions containing the purified rSdrG (50-597) were identified bydetermining the absorbance at 280 nm and by SDS-PAGE. The truncated Aregion of ClfA was purified as previously reported (17).

[0034] In accordance with the present invention, the SdrG proteins orSdrG A region from S. epidermidis may be utilized as compositions totreat or prevent thrombin-induced coagulation in human or animalpatients, and thus to treat or prevent a wide variety of conditionsassociated therewith. In the preferred composition, the SdrG protein isutilized in an amount effective to treat or prevent thrombin-inducedcoagulation, and the composition comprises the effective amount of theSdrG protein along with a pharmaceutically acceptable vehicle, carrieror excipient as would be well known to those of ordinary skill in theart including such materials as saline, dextrose, water, glycerol,ethanol, other therapeutic compounds commonly used, and combinationsthereof. As one skilled in this art would recognize, the particularvehicle, excipient or carrier used will vary depending on the nature ofthe patient and the patient's condition, and a variety of modes ofadministration would be suitable for the compositions of the invention,as would be recognized by one of ordinary skill in this art. Suitablemethods of administration of any pharmaceutical composition disclosed inthis application will preferably be intravenous, but other suitablemethods of administering a compound for the desired purpose orpreventing or reducing thrombin-induced coagulation may be introduced inother suitable ways as would be known to those of ordinary skill in thisart.

[0035] As indicated above, it is preferred that the compositions andmethods in accordance with the invention comprise SdrG or its A regionin an amount effective to prevent or reduce thrombin-induced coagulationin the blood and thus be effective in the treatment or prevention ofthrombin-induced coagulation under conditions such as venous thrombosiswherein such treatment or prevention is highly desirable. By effectiveamount is meant that level of use of the SdrG proteins of the presentinvention that will be sufficient to prevent or reduce thrombin-inducedcoagulation in accordance with the invention, and thus be useful in thetreatment or prevention of a condition wherein thrombin-inducedcoagulation is sought to be prevented or alleviated. As would berecognized by one of ordinary skill in this art, the particular amountof the SdrG protein to be used in accordance with the invention to treator prevent thrombin-induced coagulation or a condition characterizedthereby will vary depending on the nature and condition of the patient,and/or the severity of the pre-existing condition to be treated orprevented, such as in an operation wherein blood coagulation isdisfavored.

[0036] In accordance with the present invention, a method is thusprovided for treating or preventing thrombin-induced coagulation ofblood comprising administering to a human or animal patient in needthereof an SdrG protein such as the binding region A of SdrG in anamount effective to prevent or reduce thrombin-induced coagulation inthe blood. The SdrG protein may be used in the form of a therapeutic,pharmaceutically-acceptable composition as described above, the amountutilized will be the amount effective in treating or preventingthrombin-induced coagulation as also described in more detail above. Asalso indicated above, the SdrG protein utilized in the invention ispreferably a recombinant protein, and in the particularly preferredembodiment, a recombinant SdrG protein is used which constitutes theSdrG A region and which has the sequence of the residues 50-597 of SdrG.In the preferred method, the SdrG compounds and compositions of theinvention are administered in any suitable way to effect introduction ofthe active agent into the patient's bloodstream or other applicable areain order to achieve the desired goal of reducing or preventingthrombin-induced coagulation in a human or animal patient. As one ofordinary skill in this art would recognize, this can be accomplished ina number of suitable ways, including direct intravenous or intraarterialinjection, or via injection into other target areas where theanti-coagulant effects of the compositions of the invention are needed.For example, the present compositions may be utilized in the same mannerthat heparin is introduced into a patient to achieve anti-coagulationeffects, e.g., through an intravenous injection or in other suitableways. In the preferred method, the SdrG compositions are administeredfor as long as necessary to achieve the desired anti-coagulant effect aswould be determined, e.g., by the physician or other health careprofessional administering such treatment to a patient. This could beaccomplished both in the treatment of a condition wherein therapeuticanti-coagulant treatment is necessary or where preventive treatment isneeded such as in an operation wherein maximization of anti-coagulativeeffects is desired.

[0037] Accordingly, the present invention contemplates administration ofeffective amounts of the fibrinogen binding compositions of theinvention as necessary to achieve a result associated with theinhibition of thrombin-induced coagulation, such as the prevention orreduction in binding of thrombin to fibrinogen, the interference orinhibition of the release of fibrinopeptide B from fibrinogen, or thetreatment or prevention of coagulation during a disease condition suchas venous thrombosis, myocardial infarction, etc. In accordance with thepresent invention, it is contemplated that fibrinogen-binding proteinsfrom S. epidermidis may be used to prevent or treat thrombin-inducedcoagulation wherein said fibrinogen-binding proteins, such as SdrG orFbe, or their respective A domains, are capable of binding the Bβ chainof fibrinogen. More particularly, the invention contemplates thatfibrinogen-binding proteins, or their active subregions such as the Adomains from SdrG or Fbe, which can bind at the site from about residues6 to 20 on the Bβ chain of fibrinogen will be useful in methods toprevent or treat thrombin-induced coagulation and the conditionsassociated therewith. In these methods, an effective amount of thefibrinogen-binding protein is preferably administered in order toachieve the desired result of reducing or preventing thrombin-inducedcoagulation, and said fibrinogen-binding proteins may be utilized incompositions containing an effective amount of the active agent alongwith a pharmaceutical acceptable vehicle, carrier or excipient.

[0038] The examples which follow are provided which relate to certainaspects of the present invention and which exemplify certain aspects ofthe present invention. However, it will be appreciated by those of skillin the art that the techniques disclosed in the examples are onlyexemplary of techniques associated with the present invention, and thatthose of ordinary skill in the art recognize that, in light of theteachings of the present specification, many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

EXAMPLES EXAMPLE 1 Preparation of Recombinant SdrG and the Determinationof Its Binding Attributes In Accordance With the Present Invention

[0039] Summary

[0040]Staphylococcus epidermidis is an important opportunistic pathogenand is a major cause of foreign body infections. We have characterizedthe ligand-binding activity of SdrG, a fibrinogen-binding MSCRAMM fromS. epidermidis. Western ligand blot analysis showed that a recombinantform of the N-terminal A-region of SdrG bound to the native Bβ chain offibrinogen (Fg) and to a recombinant form of the Bβ chain expressed inE. coli. By analyzing recombinant truncates and synthetic peptidemimetics of the Fg Bβ chain, the binding site for SdrG was localized toresidues 6-20 of this polypeptide. Recombinant SdrG s bound to asynthetic 25 amino acid peptide (β1-25) representing the N-terminus ofthe Fg Bβ chain with a K_(D) of 1.4×10⁻⁷ M as determined by fluorescencepolarization experiments. This was similar to the apparent K_(D)(0.9×10⁻⁷ M) calculated from an ELISA where SdrG bound immobilized Fg ina concentration dependent manner. SdrG could recognize fibrinopeptide B(residues 1-14), but with a substantially lower affinity than thatobserved for SdrG binding to synthetic peptides β1-25 and β6-20.However, SdrG does not bind to thrombin digested Fg. Thus, SdrG appearsto target the thrombin cleavage site in the Fg Bβ chain. In fact, SdrGwas found to inhibit thrombin-induced fibrinogen coagulation byinterfering with fibrinopeptide B release.

[0041] Introduction

[0042] Coagulase-negative staphylococci (CNS) are importantopportunistic pathogens that are particularly associated with foreignbody infections in humans. Staphylococcus epidermidis is the most commonpathogenic species of CNS¹ and accounts for 74-92% of the infectionscaused by this group of staphylococci (1).

[0043] The molecular pathogenesis of most infections is complex andinvolves multiple microbial factors and host components, but isgenerally initiated by the adherence of the microbe to host tissues.Bacterial adherence involves specific surface components calledadhesins. Bacterial pathogens, such as staphylococci that live in theextracellular space of the host, target extracellular matrix (ECM)components, including fibrinogen (Fg) and fibronectin, for adherence andcolonization. This process is mediated by a sub-family of adhesins thathave been termed MSCRAMMs (microbial surface components recognizingadhesive matrix molecules) (2). Staphylococcus aureus expresses multipleMSCRAMMs of which several have been characterized in some detail (For arecent review see Ref. 3).

[0044] In addition to S. epidermidis, S. aureus also causes seriousforeign body infections. S. aureus appears to adhere to the biomaterialthrough an indirect mechanism. Upon implantation, the foreign bodyrapidly becomes coated with host proteins derived primarily from plasmawith Fg being a dominant component. S. aureus appears to adhere to theabsorbed proteins rather than to the biomaterial itself using adhesinsof the MSCRAMM family (4, 5). At least four of the S. aureus MSCRAMMsrecognize Fg. Two of these MSCRAMMs, clumping factor A and B (ClfA ,ClfB ), have Fg-binding A-regions followed by a long segment of Ser-Asp(SD) dipeptide repeats. The other two Fg-binding MSCRAMMs, contain asimilar ligand binding A-region followed by a fibronectin binding motifthat is repeated 5 times (6). Because the fibronectin binding activitywas identified first, these two MSCRAMMs are known as fibronectinbinding protein A and B (FnbpA and FnbpB) (7, 8). Studies havedemonstrated the importance of ClfA and ClfB in the adherence of S.aureus to plasma-coated biomaterials. S. aureus mutants deficient in oneor both of these MSCRAMMs exhibited an impaired ability to adhere toplasma-coated catheters in vivo or ex vivo (9, 10).

[0045] For S. epidermidis, adherence to foreign bodies could involveboth specific and non-specific processes. The bacteria may initiallyassociate directly with the foreign body through non-specificinteractions, while the later stages of adherence may involve morespecific interactions between bacterial adhesins and host ligands. S.epidermidis expresses polysaccharide adhesins including PS/A and PIA,which are encoded by the ica locus (11, 12). In addition, we (13) andothers (14) have recently shown that S. epidermidis contains surfaceproteins structurally related to S. aureus MSCRAMMs. Two of these S.epidermidis proteins, called SdrF and SdrG, have features typical ofGram-positive bacterial proteins that are anchored to the cell wall.Both proteins show significant amino acid sequence homology to ClfA andClfB from S. aureus including an ˜500 amino acid long A region, a SDdipeptide repeat region and features required for cell wall anchoring,including a LPXTG motif (FIG. 1A). Recent studies by Pei, et al. suggestthat an S. epidermidis protein called Fbe can bind Fg and specificallyrecognizes the Bβ chain of this molecule (15). In the current study, wehave localized the SdrG binding site in the Fg Bβ chain to theN-terminal segment of this polypeptide, proximal to the thrombincleavage site. In fact, we have demonstrated that SdrG inhibitsthrombin-induced fibrin clot formation by interfering with the releaseof fibrinopeptide B.

EXPERIMENTAL PROCEDURES

[0046] Bacterial Strains and Growth Conditions

[0047]Escherichia coli strain JM101 was used for plasmid cloning. E.coli strain Topp3 (Stratagene) was used for protein expression. Strainsharboring plasmids were grown in Lennox L broth (Sigma) or on Lennox Lagar (Sigma) supplemented with 100 μg/ml ampicillin.

[0048] PCR Amplification of the sdrG Gene Fragment

[0049] The gene fragment encoding the entire A-region was amplified byPCR using S. epidermidis K28 genomic DNA as a template. Theoligonucleotide primers used were 5′-CCCGGATCCGAGGAGAATACA GTACAAGACG-3′(SEQ ID NO: 2) and 5′-CCCGGTACCGATTTTTTCAGGAGGCAAGTCACC-3′ (SEQ ID NO:3). The restriction enzyme cleavage sites (underlined) BamHI and KpnIwere incorporated into the forward and reverse primers, respectively.The reactions were carried out using a Perkin-Elmer DNA thermocyclcer.The reactions contained 50 ng of template DNA, 100 pmol of forward andreverse primers, 20 mM Tris-HCl (pH 8.8), 2 mM MgSO₄, 10 mM KCl, 10 mM(NH₄)₂SO₄, 0.1% Triton X-100, 25 mM of each dNTP, and 5 units of Pfu DNApolymerase (Stratagene). Amplification was performed at 94° C. for 1min, 50° C. for 1 min, 72° C. for 4 min, for 25 cycles.

[0050] Cloning of sdrG into the Expression Plasmid

[0051] The amplified sdrG fragment was digested with BamHI and KpnI andligated into the expression plasmid pQE30 (Qiagen Inc.) that had beendigested with the same enzymes, yielding the construct pSdrG(50-597).The recombinant protein rSdrG(50-597) expressed from this plasmidcontains an N-terminal extension of six histidine residues (His-tag).

[0052] Expression and Purification of Recombinant MSCRAMM Protein

[0053]E. coli transformed with pSdrG(50-597) was grown for ˜2 h for thecultures to give an OD₆₀₀ of 0.6. rSdrG(50-597) expression was inducedby the addition of isopropyl-β-thiogalactopyranoside (IPTG) (Gibco-BRL)(225 μM) and the cultures were incubated at 37° C. for an additional 3h. Bacteria were pelleted and resuspended in phosphate buffered saline(PBS), pH 7.5 (140 mM NaCl, 270 μM KCl, 430 μM Na₂HPO₄, 147 μM KH₂PO₄)and frozen o/n at −20° C. Bacterial cells were thawed and mechanicallylysed by using a French Pressure Cell (SLM Amnico). Cell debris wasremoved by centrifugation and filtration through a 0.45 μm filtermembrane. The supernatant containing the recombinant protein was appliedto a Ni²⁺ charged (87.5 mM) 5 ml Hi Trap chelating column (AmershamPharmacia Biotech) connected to a FPLC system. The column wasequilibrated With buffer A (0.1 M NaCl, 10 mM Tris-HCl, pH 8.0) beforethe application of the filtered lysate. The column was then washed with10 bed volumes of buffer A containing 5 mM imidazole. Bound protein waseluted with a continuous linear gradient of imidazole (5-120 mM; totalvolume 160 mls) in buffer A. Fractions were monitored for protein bydetermining the absorbance at 280 nm and fractions containingrSdrG(50-597) were identified by SDS-PAGE (16). These fractions werepooled and dialyzed against PBS, pH 7.5. The dialyzed protein was thenapplied to a Q-Sepharose column (Amersham Pharmacia Biotech)equilibrated with 25 mM Tris-HCl, pH 8.0. Bound protein was eluted witha continuous linear gradient of NaCl (0-0.5 mM; total volume 160 mls) in25 mM Tris-HCl, pH 8.0. Fractions containing the purified rSdrG(50-597)were identified by determining the absorbance at 280 nm and by SDS-PAGE.The truncated A region of ClfA was purified as previously reported (17).

[0054] Synthetic Peptides

[0055] The synthetic Fg peptides β1-25, β1-25S, β1-20, β6-25, werecustom ordered from Research Genetics and the fibrinopeptides A and B(FpA and FpB) were from Bachem. Peptides β6-20 and β11-20 weresynthesized in our laboratory using a multiple peptide synthesizer byAdvanced Chemtech. For the following peptides the residue numbers aregiven and the sequence follows (Residue 1 corresponds to the firstresidue of the mature Bβ chain.): peptide β1-25, is composed of thefirst 25 amino acid residues of the N-terminus of the Bβ chain of Fg(QGVNDNEEGFFSARGHRPLDKK REE) (SEQ ID NO: 4), peptide β1-20(QGVNDNEEGFFSARGHRPLD) (SEQ ID NO: 5), peptide β6-25(NEEGFFSARGHRPLDKKREE) (SEQ ID NO: 6), peptide β1-25S is a scrambledversion of peptide β1-25 (FSERKDLHQGEGNPREFVENDAKGR) (SEQ ID NO: 7),peptide β6-20 (NEEGFFSA RGHRPLD) (SEQ ID NO: 8), peptide β11-20(FSARGHRPLD) (SEQ ID NO: 9), FpA (ADSEGEGDFLAEGGGVR) (SEQ ID NO: 10),and FpB (QGVNDNEEGFFSAR) (SEQ ID NO: 11). Peptides were purified by HPLCand analyzed by MALDI mass spectrometry.

[0056] ELISA

[0057] Microtiter plates (Immulon 4, Dynatech Laboratories Inc.) werecoated with 1 μg of Fg (Enzyme Research Labs) in PBS, pH 7.5 for 18 h at4° C. Plates were washed three times with PBS, 0.05% Tween 20 (PBST) andblocked with 1% (w/v) bovine serum albumin (BSA) for 1 h at roomtemperature. Plates were washed three times with PBST and rSdrG(50-597),diluted into PBS, was added to the wells and the plate was incubated for1 h at room temperature. Plates were washed three times with PBST andbound rSdrG(50-597) was detected by adding a 1:2000 dilution of ananti-His-tag mAb (Clontech) in PBST, 0.1% BSA. Plates were incubated for1 h at room temperature and then washed three times with PBST. A 1:2000dilution of goat anti-mouse alkaline phosphatase (AP)-conjugatedpolyclonal antibodies (Bio-Rad) in PBST, 0.1% BSA were added to thewells and the plate was incubated for 1 h at room temperature. Plateswere washed three times with PBST and developed with p-nitrophenylphosphate (Sigma) in 1 M diethanolamine, 0.5 mM MgCl₂, pH 9.0 at roomtemperature for ˜30 min. Plates were read at 405 nm using an ELISA platereader (Thermomax microplate reader, Molecular Devices).

[0058] In the inhibition experiments, 50 nM rSdrG(50-597) in PBS waspre-incubated with the indicated amounts of selected peptides for 1 h atroom temperature. The sample mixtures were added to the Fg-coated wellsand bound rSdrG(50-597) was detected as described above.

[0059] For the ELISA with thrombin-digested Fg, the plate was coatedwith Fg and blocked as described above. The plate was washed three timeswith PBST and 50 μl of 1.0 NIH unit/ml of thrombin was added to the Fgcoated wells. The plate was incubated at 37° C. for 30 min. The platewas washed three times with PBST and 50 μl of 1.0 NIH unit/ml of hirudin(Sigma) was added to the wells and incubated at 37° C. for 30 min. Theplate was washed three times with PBST and blocked with 1% BSA for 1 hat room temperature. After washing three times with PBST, 100 μl ofbiotin labeled rSdrG(50-597) (25-1000 nM) or a rSdrG(50-597)/hirudin(1.0 NIH unit/ml) mixture was added to the wells and incubated for 1 hat room temperature. The plate was washed three times with PBST and a1:5000 dilution of streptavidin-AP conjugated (Boehringer Mannheim) inPBST/0.1% BSA was added to the wells for 1 h at room temperature. Theplate was washed three times with PBST and developed as described above.

[0060] Construction of Fg Bβ Chain Truncates

[0061] An E. coli strain harboring plasmid p668 which contains the cDNAfor the Fg Bβ chain was kindly provided by Dr. Susan T. Lord (Universityof North Carolina, Chapel Hill, N.C.). The 1525 bp fragment from p668was subcloned into the plasmid pQE30 to produce recombinant mature Bβchain with a N-terminal His-tag. Additional Bβ chain constructs (FIG.3A) were made by subcloning into either pQE30 or pGEX-KG (Pharmacia) toproduce recombinant proteins with a N-terminal His-tag or GlutathioneS-transferase (GST) fusion.

[0062] Western Ligand Blot Analysis

[0063] Whole E. coli lysates harboring each respective Fg Bβ chainconstruct were fractionated by SDS-PAGE and the separated proteins weretransferred to nitrocellulose membrane with a semi-dry transfer cell(Bio-Rad). The membrane was incubated overnight with 5% (w/v) non-fatdry milk in PBS, pH 7.5 at 4° C. to saturate non-specific binding sites.After blocking, the membrane was washed three times with PBST and thenincubated with biotin labeled rSdrG(50-597) (0.5 μM) for 1 h at roomtemperature. rSdrG(50-597) was biotin labeled using EZ Link-sulfo-NHS-LCbiotin (Sigma) according to the manufacturers' instructions. After threemore washes with PBST, the blot was incubated with a 1:5000 dilution ofstreptavidin-AP conjugated for 1 h at room temperature and developedwith 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro bluetetrazolium (NBT) (Biorad) in carbonate:bicarbonate buffer (14 mMNa₂CO₃, 36 mM NaHCO₃, 5 mM MgCl₂:6H₂O, pH 9.8) for ˜15 min at roomtemperature.

[0064] Fluorescence Polarization

[0065] Fluorescence polarization was used to determine the equilibriumdissociation constant (K_(D)) for the interaction of rSdrG(50-597) withpeptide β1-25. The peptide was labeled with fluorescein as previouslydescribed (18). Increasing concentrations of rSdrG(50-597) in PBS, pH7.5 , were incubated with 10 nM labeled peptide for 3 h in the dark atroom temperature. Reactions were allowed to reach equilibrium.Polarization measurements were taken with a Luminescence SpectrometerLS50B (Perkin Elmer) using FL WinLab software (Perkin Elmer). Bindingdata was analyzed by nonlinear regression used to fit a binding functionas defined by the following equation: $\begin{matrix}{{\Delta \quad P} = \frac{\Delta \quad {P_{\max} \cdot \lbrack{protein}\rbrack}}{K_{D} + \lbrack{protein}\rbrack}} & \text{Equation~~1}\end{matrix}$

[0066] where ΔP corresponds to the change in fluorescence polarization,ΔP_(max) is the maximum change in fluorescence, and K_(D) is theequilibrium dissociation constant of the interaction. A single bindingsite was assumed in this analysis.

[0067] Fg Coagulation Assay

[0068] 150 μl of a 3.0 μM Fg solution was incubated with 10 μl ofrSdrG(50-597) or BSA (1.0-6.0 μM) and 50 μl of thrombin (Sigma) (1.0 NIHunit/ml) in microtiter wells at room temperature. Clot formation wasmonitored by measuring the increase in optical density (OD) at 405 nmover time and expressed as V_(max) (mOD/min). A plate reader (Thermomaxmicroplate reader, SOFTmax software, Molecular Devices) was used tomonitor OD. Using the kinetic mode with one wavelength (L1=405 nm),samples were read every 10 sec for 5 min. In this assay, 1.0 NIH unit/mlof thrombin incubated with 3.0 μM Fg produced a fibrin clot in 5 min atroom temperature.

[0069] Release of Fibrinopeptides by Thrombin

[0070] The thrombin catalyzed release of fibrinopeptides A and B wasanalyzed as follows. Fg solutions were diluted to 0.3 μM in 20 mM HEPES(pH 7.4), 150 mM NaCl, 5 mM ε-aminocapriotic acid, and 1.0 mM CaCl₂.ε-aminocapriotic acid was included to inhibit any possible plasmincontaminant activity. Thrombin was added to a final concentration of0.05 NIH units/ml. rSdrG(50-597) was added to a final concentration of0.3 μM, 0.6 μM, or 1.5 μM. The tubes were mixed by inversion and 500 μlaliquots were removed for the 15 and 60 min time points. The aliquotswere incubated at room temperature and then immersed in boiling waterfor 15 min to halt the reaction. The aliquots were stored on ice for theremainder of the time course. At the end of the reaction the sampleswere centrifuged for 15 min at 4° C., and the supernatants were removedand stored at −20° C. overnight prior to analysis by high performanceliquid chromatography.

[0071] The fibrinopeptides released were monitored by reverse phase HPLCessentially as described (19). The samples were loaded onto a WatersDelta-Pak C₁₈ column equilibrated with buffer A (25 mM NaH₂PO₄/Na₂HPO₄,pH 6.0). Fibrinopeptides were eluted with a linear gradient from 100%buffer A to 40% buffer B (Buffer A with 50% acetonitrile) and monitoredby absorbance at 205 nm. Fibrinopeptide peak area was determined usingthe software Waters Millennium³².

RESULTS

[0072] Expression and Purification of Recombinant SdrG A-region

[0073] In order to characterize the ligand binding activity of SdrG, arecombinant form of the putative ligand binding A-region (residues50-597) (FIG. 1C) was expressed in E. coli with a N-terminal His-tag.This protein construct, rSdrG(50-597), was purified by metal chelateaffinity chromatography followed by ion-exchange chromatography. Thepurity of the recombinant protein was confirmed by SDS-PAGE analysis,where it migrated with an apparent molecular mass of ˜97 kDa (FIG. 1B).This is larger than the theoretical molecular weight of 63.7 kDapredicted from the primary amino acid sequence of this protein. Analysisof rSdrG(50-597) by MALDI mass spectrometry indicated a molecular massof 63.3 kDa. Aberrant migration in SDS-PAGE has also been observed withrecombinant MSCRAMMs derived from S. aureus, and may be explained by thehydrophilic nature of these proteins (9, 13).

[0074] SdrG Binds the Fg Bβ Chain

[0075] SdrG is closely related to the recently described fibrinogenbinding MSCRAMM Fbe (15). Therefore, we initially examined the ligandbinding specificity of SdrG for Fg in an ELISA. In this assay,rSdrG(50-597) bound immobilized Fg, but failed to bind to otherimmobilized ECM proteins such as fibronectin, collagen types I and IV,vitronectin, laminin and thrombospondin (data not shown). Binding ofincreasing concentrations of rSdrG(50-597) to absorbed Fg exhibitedsaturation kinetics (FIG. 2). Together these observations demonstratethe specificity of the SdrG-Fg interaction. Furthermore, biotin labeledrSdrG(50-597) recognized the Bβ chain, but not the Aα or γ chains of Fgwhen analyzed by Western ligand blotting (data not shown).

[0076] Localization of the SdrG Binding Site in the Fg Bβ Chain

[0077] The observation that rSdrG binds the Fg Bβ chain fractionatedunder reducing and denaturing conditions in Western ligand blot analysissuggests that the MSCRAMM recognizes a specific linear amino acidsequence in the Bβ chain. To explore this possibility and locate theSdrG binding site, a recombinant mature Fg Bβ chain and a series oftruncated forms of the Bβ chain expressed in E. coli were analyzed byWestern ligand blot. The recombinant Bβ chain constructs were expressedas either His-tag or glutathione S-transferase (GST) fusion proteins(FIG. 3A). The fractionated proteins were transferred to a supportingmembrane and probed with biotin labeled rSdrG(50-597) (FIG. 3B).rSdrG(50-597) recognized the mature recombinant Bβ chain (residues1-462) as well as the recombinant truncates encompassing residues 1-341,1-220, 1-195 and 1-95. However, rSdrG(50-597) failed to bind to the tworecombinant truncates that lacked the N-terminal 25 amino acid residuesof the Bβ chain, rβ(25-95) and rβ(25-195) (FIG. 3B). These observationsdemonstrate that rSdrG(50-597) recognizes a linear sequence in Fg andsuggests that this site lies within the N-terminal region of the Bβchain.

[0078] Inhibition of rSdrG(50-597) Binding to Fg by Synthetic Peptides

[0079] To further define the rSdrG(50-597) binding site in the Fg Bβchain, we used a peptide mimetic approach. A series of peptidesrepresenting segments of the N-terminal region of the FgBβ chain weresynthesized and tested for their ability to inhibit the binding ofrSdrG(50-597) to Fg in an ELISA (FIG. 4). In FIG. 4A, peptides β1-25 andβ6-25 were shown to inhibit the binding of rSdrG(50-597) to Fg in aconcentration dependent manner, whereas the scrambled version of β1-25,peptide β1-25S, did not interfere with the binding of rSdrG(50-597) toFg. Effective inhibition of rSdrG(50-597) binding to Fg was alsoobserved with peptide β6-20 and, to a somewhat lesser degree, withβ1-20. Peptide β11-20 was essentially inactive in this assay (FIG. 4B).

[0080] The thrombin cleavage sites in Fg lie between residues 14 (Arg)and 15 (Gly) in the Bβ chain and between 16 (Arg) and 17 (Gly) in the Aαchain. Upon cleavage of Fg by thrombin the fibrinopeptides, FpA and FpB,are sequentially released. The fibrinopeptides were examined asinhibitors of rSdrG(50-597) binding to Fg in an ELISA. FpB inhibited thebinding of rSdrG(50-597) in a concentration dependent manner, but thispeptide was at least 10 fold less active than the synthetic peptideβ1-25 (FIG. 4C). FpA was essentially inactive and behaved similar to thescrambled peptide β1-25S. Taken together, this suggest thatrSdrG(50-597) recognizes a linear amino acid sequence in the Bβ chainlocated within residues 6-20. This recognition site appears to overlapthe thrombin cleavage site in this polypeptide.

[0081] rSdrG Binding to Thrombin-Digested Fg

[0082] The rSdrG binding site seems to lie within close proximity to thethrombin cleavage site, therefore, we investigated if rSdrG(50-597)could bind to Fg in which the thrombin cleavage site was abolished. Fgcoated microtiter wells were pretreated with thrombin or thrombin plushirudin (which inhibits thrombin activity) in order to remove FpB anddestroy the cleavage site. The ability of rSdrG(50-597) to bind to thisthrombin digested Fg was significantly impaired (FIG. 5), suggestingthat the thrombin cleavage site residues Bβ 14 (Arg), 15 (Gly) andresidues within FpB (1-14) are essential for rSdrG(50-597) to bind Fg.

[0083] Determination of Equilibrium Dissociation Constants (K_(D))

[0084] An equilibrium dissociation constant (K_(D)) for the interactionof rSdrG(50-597) with the Fg Bβ chain peptide β1-25 was determined. Byanalyzing the binding of increasing concentrations of rSdrG(50-597) tothe fluorescein-labeled β1-25 peptide in a fluorescence polarizationassay, rSdrG(50-597) binding to the labeled peptide exhibited saturationkinetics with a K_(D) of 1.4±0.01×10⁻⁷ M (FIG. 6A). To demonstrate thespecificity of this interaction, the binding of rSdrG(50-597) to thelabeled β1-25 peptide was measured in the presence of increasing amountsof unlabeled peptide (β1-25) or scrambled peptide (β1-25S). Theunlabeled β1-25 peptide, but not peptide β1-25S inhibited binding ofrSdrG(50-597) to the fluorescein-labeled β1-25 peptide, in aconcentration dependent manner (FIG. 6B). The apparent K_(D) determinedfor the binding of rSdrG(50-597) to the fluorescein labeled peptideβ1-25 is similar to the apparent K_(D) (0.9×10⁻⁷ M) for the interactionof rSdrG(50-597) with immobilized, intact Fg as determined by ELISA(FIG. 2).

[0085] rSdrG(50-597) Inhibits Thrombin-Induced Fibrin Clot Formation

[0086] In the final stages of the blood coagulation cascade, thrombincleaves Fg releasing the fibrinopeptides and producing fibrin monomers.These fibrin monomers then polymerize to form a fibrin clot (20). Thelocalization of the SdrG binding site described above raises thepossibility that rSdrG(50-597) may be able to inhibit thrombin-inducedfibrin clot formation, perhaps by directly competing with thrombin forbinding to the N-terminus of the Bβ chain of Fg or by binding to aproximal site and sterically blocking thrombin's proteolytic attack onthe Bβ chain. To test this hypothesis, we designed a fibrin clotinhibition assay in which 3.0 μM Fg, 0-6.0 μM rSdrG(50-597) and 1.0 NIHunit/ml of thrombin were incubated and the formation of a fibrin clotwas monitored by measuring the increase in optical density at 405 nm.FIG. 7 shows that rSdrG(50-597) inhibited fibrin clot formation in aconcentration dependent manner, whereas BSA had no effect. This suggeststhat rSdrG(50-597) can interfere with thrombin activity by binding to asite in the Fg Bβ chain that is proximal to or overlaps the binding sitefor thrombin.

[0087] Analysis of Fibrinopeptide B Release by HPLC

[0088] The release of FpA and FpB from the N-terminus of the Aα and Bβchains of Fg by thrombin can be monitored and quantitated by highperformance liquid chromatography (19, 21, 22). We examined the effectof rSdrG(50-597) on fibrinopeptide release by measuring the peak areasof FpA and FpB, as detected by HPLC. The HPLC chromatograms shown inFIG. 8 show the expected fibrinopeptide release following digestion ofFg with thrombin superimposed with the fibrinopeptide release when Fgand thrombin are incubated with rSdrG(50-597). A significant decrease inthe amount of FpB release was shown with a 1:1 ratio of rSdrG(50-597) toFg (Table I) whereas, a 5:1 ratio was effectively able to inhibit therelease of FpB (FIG. 8). This effect was seen at an incubation time of15 min and 60 min. There was no apparent interference of FpA release byrSdrG(50-597). TABLE I Percentage of FpB released in the presence ofSdrG Eg (0.3 μM) was incubated with SdrG and 0.5 NIH units/ml ofthrombin at room temperature and the samples were analyzed by HPLC. Theamount of fibrinopeptide released was determined by measuring the areaunder the peaks on the HPLC chromatograms. The data was normalized inorder to compare the data from separate chromatograms assuming that therelease of FpA is not affected by the presence of SdrG. The peak arearepresenting FpB in the absence of SdrG was set to 100%. SdrG:Fg 15 min60 min 0:1 100 100 1:1 65.4 45.2 2:1 13.9 17.4 5:1 <0.001 <0.001

DISCUSSION

[0089] In this study, we have shown that SdrG binds the N-terminus ofthe Bβ chain of Fg with a high degree of specificity. The binding ofSdrG to an N-terminal Fg peptide exhibits a K_(D) of 1.4×10⁻⁷ M, whichis significantly lower than the K_(D) determined for the binding of ClfAto a γ chain peptide (2.0×10⁻⁵ M) (18). Thus, SdrG appears to have ahigher affinity for its respective synthetic Fg peptide target comparedto the S. aureus MSCRAMM. The K_(D) determined for the binding of SdrGto the synthetic peptide β1-25 is similar to the apparent K_(D)estimated for the binding of SdrG to intact Fg absorbed onto microtiterwells. This observation suggests that the SdrG binding site in thesynthetic peptide is presented in a nearly optimal form and thatadditional segments of Fg do not significantly contribute to theformation of the SdrG binding site.

[0090] Several studies have examined the role of Fg binding MSCRAMMsfrom S. aureus as virulence factors in animal models. Strains in whichthe genes encoding ClfA or ClfB have been inactivated are less virulentcompared to the wild type strain in a rat model of catheter-inducedendocarditis (23, 24). These results suggest that ClfA- andClfB-mediated adherence is required for the maximum virulence potentialof S. aureus to be expressed. ClfB has been shown to promote S. aureusadherence to ex vivo hemodialysis tubing, further confirming that ClfBcontributes to bacterial attachment to biomaterials coated with hostproteins (9). In a recent study, Stutzmann Meier, et al. showed thatheterologous expression of ClfA on Streptococcus gordonii, which isgenerally considered a non-virulent bacterium, rendered this organismpathogenic in a rat endocarditis model (25). With the discovery thatSdrG is a Fg binding MSCRAMM expressed by S. epidermidis, thepossibility arises that SdrG can act as a virulence factor in S.epidermidis-induced infections and plays a role similar to that of theFg binding MSCRAMMs in S. aureus-induced infections.

[0091] We have mapped the binding site of rSdrG(50-597) in the Fg Bβchain to a linear sequence in the N-terminal region of this polypeptide.Peptide β6-20 is a potent inhibitor of the binding of rSdrG(50-597) toFg, whereas FpB (1-14) has poor inhibitory activity. Because peptideβ6-20, but not β11-20 is recognized by this MSCRAMM, the N-terminalborder of the binding site must lie between residues 6 and 11 of the Bβchain. The observation that rSdrG(50-597) is unable to bind to thrombindigested Fg, i.e. the fibrinopeptides are absent, suggests that theC-terminus of this binding site is located between residues 14 and 20 ofthe Bβ chain.

[0092] It is striking that many of the identified staphylococcalMSCRAMMs appear to specifically recognize Fg, although the sitestargeted in Fg by these proteins vary. ClfA , FnbpA and FnbpB of S.aureus all recognize the C-terminus of the Fg γ chain (6, 27). ClfB fromS. aureus targets an as yet unidentified site in the Aα chain (9) andSdrG is here shown to bind to the N-terminus of the Bβ chain. Thus,these MSCRAMMs use a conserved A region to bind different sites in Fg.Furthermore, the MSCRAMMs appear to target sites in Fg that areimportant in the molecular physiology of this key component ofhemostasis. The C-terminus of the γ chain is recognized by the plateletintegrin α_(IIb)β₃ and ClfA is a potent inhibitor of Fg-induced plateletaggregation (26, 27). Here, we show that the binding site in the Bβchain for rSdrG(50-597) appears to overlap the thrombin cleavage siteand that rSdrG(50-597) can interfere with fibrin clot formation byinhibiting the thrombin-induced release of FpB. Fg may play an importantrole in the host's defense against microbial infections and interferingwith this function gives the bacteria an advantage and the ability tosurvive in a hostile environment. One such potential advantage may berelated to the observed chemotactic activity of FpB for human peripheralblood leukocytes (28-30). We have shown that rSdrG(50-597) can preventthe release of FpB, thus one can speculate that the reason S.epidermidis possesses a protein that can bind to this region of the FgBβ chain is to prevent the release of chemotactic elements. This mayreduce the influx of phagocytic neutrophils and help to ensure thesurvival of the bacteria in the host.

REFERENCES

[0093] The following articles are incorporated herein by reference:

[0094] 1. Garrett, D. O., Jochimsen, E., Murfift, K., Hill, B.,McAllister, S., Nelson, P., Spera, R. V., Sall, R. K., Tenover, F. C.,Johnston, J., Zimmer, B., and Jarvis, W. R. (1999) Infect Control HospEpidemiol 20(3), 167-70.

[0095] 2. Patti, J. M., and Höök, M. (1994) Curr Opin Cell Biol 6(5),752-8

[0096] 3. Foster, T. J., and Höök, M. (1998) Trends Microbiol 6(12),484-8

[0097] 4. Galliani, S., Viot, M., Cremieux, A., and Van der Auwera, P.(1994) J Lab Clin Med 123(5), 685-92.

[0098] 5. Vaudaux, P., Pittet, D., Haeberli, A., Huggler, E., Nydegger,U. E., Lew, D. P., and Waldvogel, F. A. (1989) J Infect Dis 160(5),865-75.

[0099] 6. Wann, E. R., Gurusiddappa, S., and Höök, M. (2000) J Biol Chem275(18), 13863-71.

[0100] 7. Flock, J. I., Fröman, G., Jonsson, K., Guss, B., Signas, C.,Nilsson, B., Raucci, G., Höök, M., Wadstrom, T., and Lindberg, M. (1987)Embo J 6(8), 2351-7.

[0101] 8. Fröman, G., Switalski, L. M., Speziale, P., and Höök, M.(1987) J Biol Chem 262(14), 6564-71

[0102] 9. Ni Eidhin, D., Perkins, S., Francois, P., Vaudaux, P., Höök,M., and Foster, T. J. (1998) Mol Microbiol 30(2), 245-57

[0103] 10. Vaudaux, P. E., Francois, P., Proctor, R. A., McDevitt, D.,Foster, T. J., Albrecht, R. M., Lew, D. P., Wabers, H., and Cooper, S.L. (1995) Infect Immun 63(2), 585-90

[0104] 11. McKenney, D., Hubner, J., Muller, E., Wang, Y., Goldmann, D.A., and Pier, G. B. (1998) Infect Immun 66(10), 4711-20

[0105] 12. Cramton, S. E., Gerke, C., Schnell, N. F., Nichols, W. W.,and Götz, F. (1999) Infect Immun 67(10), 5427-33.

[0106] 13. McCrea, K. W., Hartford, O., Davis, S., Ni Eidhin, D., Lina,G., Speziale, P., Foster, T. J., and Höök, M. (2000) Microbiology 146(Pt7), 1535-46.

[0107] 14. Nilsson, M., Frykberg, L., Flock, J. I., Pei, L., Lindberg,M., and Guss, B. (1998) Infect Immun 66(6), 2666-73

[0108] 15. Pei, L., Palma, M., Nilsson, M., Guss, B., and Flock, J. I.(1999) Infect Immun 67(9), 4525-30

[0109] 16. Laemmli, U. K. (1970) Nature 227, 680-685

[0110] 17. McDevitt, D., Francois, P., Vaudaux, P., and Foster, T. J.(1995) Mol Microbiol 16(5), 895-907.

[0111] 18. O'Connell, D. P., Nanavaty, T., McDevitt, D., Gurusiddappa,S., Höök, M., and Foster, T. J. (1998) J Biol Chem 273(12), 6821-9.

[0112] 19. Mullin, J. L., Gorkun, O. V., Binnie, C. G., and Lord, S. T.(2000) J Biol Chem 275(33), 25239-46.

[0113] 20. Herrick, S., Blanc-Brude, O., Gray, A., and Laurent, G.(1999) Int J Biochem Cell Biol 31(7), 741-6

[0114] 21. Ng, A. S., Lewis, S. D., and Shafer, J. A. (1993) MethodsEnzymol 222, 341-58

[0115] 22. Haverkate, F., Koopman, J., Kluft, C., D'Angelo, A.,Cattaneo, M., and Mannucci, P. M. (1986) Thromb Haemost 55(1), 131-5.

[0116] 23. Moreillon, P., Entenza, J. M., Francioli, P., McDevitt, D.,Foster, T. J., Francois, P., and Vaudaux, P. (1995) Infect Immun 63(12),4738-43

[0117] 24. Entenza, J. M., Foster, T. J., Ni Eidhin, D., Vaudaux, P.,Francioli, P., and Moreillon, P. (2000) Infect Immun 68(9), 5443-6.

[0118] 25. Stutzmann Meier, P., Entenza, J. M., Vaudaux, P., Francioli,P., Glauser, M. P., and Moreillon, P. (2001) Infect Immun 69(2),657-664.

[0119] 26. Farrell, D. H., Thiagarajan, P., Chung, D. W., and Davie, E.W. (1992) Proc Natl Acad Sci U S A 89(22), 10729-32.

[0120] 27. McDevitt, D., Nanavaty, T., House-Pompeo, K., Bell, E.,Turner, N., McIntire, L., Foster, T., and Höök, M. (1997) Eur J Biochem247(1), 416-24

[0121] 28. Kay, A. B., Pepper, D. S., and McKenzie, R. (1974) Br JHaematol 27(4), 669-77.

[0122] 29. Richardson, D. L., Pepper, D. S., and Kay, A. B. (1976) Br JHaematol 32(4), 507-13.

[0123] 30. Senior, R. M., Skogen, W. F., Griffin, G. L., and Wilner, G.D. (1986) J Clin Invest 77(3), 1014-9.

FOOTNOTES

[0124]¹The abbreviations used are: CNS, coagulase-negativestaphylococci, ECM, extracellular matrix, Fg, fibrinogen, MSCRAMM,microbial surface component recognizing adhesive matrix molecules, ClfAand ClfB , clumping factors A and B, FnbpA and FnbpB,fibronectin-binding proteins A and B, SdrF and SdrG, serine-aspartaterepeat proteins F and G, FpA and FpB, fibrinopeptides A and B, ELISA,enzyme-linked immunosorbent assay, HPLC, high performance liquidchromatography, PCR, polymerase chain reaction, PAGE, polyacrylamide gelelectrophoresis, K_(D), equilibrium dissociation constant.

EXAMPLE 2 Experiments Showing SdrG Inhibits Serine Protease Digestion ofHuman Fibrinogen

[0125] The localization of the binding site for SdrG in Fg revealed thatthe thrombin cleavage site was in close proximity to the SdrG bindingsite. We were able to show that SdrG could not bind to thrombin-treatedFg but could, in fact, inhibit thrombin-catalyzed fibrin clot formation.In a related study, we analyzed SdrG for its ability to bind toimmobilized Fg that had been treated with other serine proteasesisolated from snake venom and for its ability to inhibit clot formationin the presence of these same proteases. The fibrinolytic activities ofsnake venoms have been well documented and their activities are similarto thrombin, however, some may be specific for the Aα chain or the Bβchain only. In this study we employed three proteases from snake venomsthat have different activities. Ancrod, an α-fibrinogenase isolated fromCalloselasma rhodostoma (Malayan Pit viper), releases only FpA and leadsto the formation of an unstable fibrin clot (Bell 1997). Contortrixobin,a β-fibrinogenase isolated from Agkistrodon contortrix contortrix(Southern Copperhead) preferentially releases FpB but does not form aclot effectively because FpA has not been released.

[0126] Results

[0127] SdrG Binding to Serine Protease-treated Immobilized Fg

[0128] In an ELISA similar to the ELISA experiment described previously,we found that rSdrG(50-597) could not bind to Fg treated with thrombinor contortrixobin, the β-fibrinogenase but could bind to untreated Fg.rSdrG(50-597) could actually bind better to Fg treated with ancrod, theα-fibrinogenase, than to untreated Fg (FIG. 9). The A□ chain mayinterfere with rSdrG(50-597) binding to the Bβ chain and thisobservation may be due to the absence of FpA, thus making the N-terminusof the Bβ chain more easily accessible.

[0129] Analysis of Fibrinopeptide B Release by HPLC

[0130] As in chapter two, we monitored the fibrinopeptide release in thepresence of SdrG and the β-fibrinogenase contortrixobin. The HPLCchromatograms shown in FIG. 10 show the expected fibrinopeptide releasefollowing digestion of Fg with contortrixobin superimposed with thefibrinopeptide release when Fg and contortrixobin are incubated withrSdrG(50-597). A decrease in the amount of FpB release is seen when a1:5 ratio of Fg to rSdrG (50-597) is used. The amount of FpB releasedwith contortrixobin is significantly less than the amount released withthrombin. This is most likely because FpA is still intact on the Fgmolecule, thus contortrixobin is not as effective at cleaving Fg due tosteric hindrance from FpA.

[0131] Discussion

[0132] The results of this study with serine proteases isolated fromsnake venoms corroborate the results seen with SdrG and thrombin.rSdrG(50-597) was not able to bind to Fg that was treated with adifferent protease that preferentially cleaves the FpB from the Bβchain. This eliminates any concern that the effect seen withthrombin-treated Fg was in any way due to thrombin inhibitingrSdrG(50-597) from binding. Because rSdrG(50-597) was still able to bindto ancrod-treated Fg this also confirms that the FpA from the Aα chainwas not involved in SdrG binding to Fg.

[0133] rSdrG(50-597) was able to effectively inhibit FpB release in thepresence of the β-fibrinogenase contortrixobin supporting the evidencethat was shown previously and substantiating the mechanism by which SdrGis inhibiting clot formation is in fact by inhibiting FpB release. Wewere, however, unable to perform the clot inhibition experiment shown inchapter two due to the ineffective clot formation when only FpB isreleased, thus there was no accurate control.

[0134] Interestingly, ancrod has been used clinically as anantithrombotic agent for a number of different disease conditionsincluding stroke, myocardial infarction, sickle-cell crisis and venousthrombosis¹ (Forbes 1993; Atkinson 1997) (Gilles, Reid et al. 1968),(Davies, Merrick et al. 1972). This defibrinating enzyme cleaves FpA,but not FpB from Fg to form a clot that is very sensitive to endogenousfibrinolysis, additionally ancrod activates plasminogen furthercontributing to fibrinolysis (Pizzo, Schwartz et al. 1972; Carr 1975;Bell 1997). The result of administering ancrod is a significantreduction in plasma Fg concentration within minutes and within hours thelevel of Fg is markedly depressed. Hypofibrinogenemia is sustained byadministering ancrod daily. After termination of treatment, the plasmaFg rises gradually, returning to normal levels in days (Bell, Bolton etal. 1968). The limited clinical experience indicates that defibrinationis achieved with ancrod with reasonable safety, however, the elaborationof neutralizing antibodies with repeated injections of ancrod leads toresistance (Pitney, Holt et al. 1969; Pitney and Regoeczi 1970),(Vinazzer 1973; Sapru, Moza et al. 1975).

[0135] SdrG inhibits clot formation by preventing the release of FpB.Although the mechanism of thrombosis prevention by SdrG is differentthan that of ancrod, the ability of SdrG to inhibit clot formation couldpotentially lead to its use as a novel anti-thrombotic agent. Certainly,much more research would be needed to reliably assess its effectivenessand safety relative to heparin. Heparin is the standard treatment forthrombotic disorders and its mode of action is by increasing theeffectiveness of anti-thrombin III. A potentially important indicationfor ancrod and possibly SdrG may be to avoid heparin-inducedthrombocytopenia, resulting from heparin treatment. In addition, sinceSdrG binds to the Bβ chain of Fg, it may be possible to also use it inconjunction with other agents, such as ancrod, which target the Aα chainof Fg in order to further enhance the anti-coagulation when necessary.If used in this fashion, in addition to a composition of SdrG used toreduce or prevent thrombin-induced coagulation, ancrod in an amounteffective to interfere or inhibit the release of fibrinopeptide A fromfibrinogen may also be administered along with the SdrG.

[0136] All of the references disclosed herein are incorporated byreference.

[0137]¹Definitions: venous thrombosis—the presence of a blood clotwithin a vein, hypofibrinogenemia—abnormal deficiency of fg in theblood, thrombocytopenia—persistent decrease in the number of bloodplatelets

1 11 1 5 PRT Staphylococcus epidermidis MISC_FEATURE (3)..(3) X = anyamino acid 1 Leu Pro Xaa Thr Gly 1 5 2 31 DNA Staphylococcus epidermidis2 cccggatccg aggagaatac agtacaagac g 31 3 33 DNA Staphylococcusepidermidis 3 cccggtaccg attttttcag gaggcaagtc acc 33 4 25 PRTStaphylococcus epidermidis 4 Gln Gly Val Asn Asp Asn Glu Glu Gly Phe PheSer Ala Arg Gly His 1 5 10 15 Arg Pro Leu Asp Lys Lys Arg Glu Glu 20 255 20 PRT Staphylococcus epidermidis 5 Gln Gly Val Asn Asp Asn Glu GluGly Phe Phe Ser Ala Arg Gly His 1 5 10 15 Arg Pro Leu Asp 20 6 20 PRTStaphylococcus epidermidis 6 Asn Glu Glu Gly Phe Phe Ser Ala Arg Gly HisArg Pro Leu Asp Lys 1 5 10 15 Lys Arg Glu Glu 20 7 25 PRT Staphylococcusepidermidis 7 Phe Ser Glu Arg Lys Asp Leu His Gln Gly Glu Gly Asn ProArg Glu 1 5 10 15 Phe Val Glu Asn Asp Ala Lys Gly Arg 20 25 8 15 PRTStaphylococcus epidermidis 8 Asn Glu Glu Gly Phe Phe Ser Ala Arg Gly HisArg Pro Leu Asp 1 5 10 15 9 10 PRT Staphylococcus epidermidis 9 Phe SerAla Arg Gly His Arg Pro Leu Asp 1 5 10 10 17 PRT Staphylococcusepidermidis 10 Ala Asp Ser Glu Gly Glu Gly Asp Phe Leu Ala Glu Gly GlyGly Val 1 5 10 15 Arg 11 14 PRT Staphylococcus epidermidis 11 Gln GlyVal Asn Asp Asn Glu Glu Gly Phe Phe Ser Ala Arg 1 5 10

What is claimed is:
 1. A method of treating or preventingthrombin-induced coagulation of blood comprising administering to ahuman or animal patient in need thereof an SdrG protein in an amounteffective to treat or prevent thrombin-induced coagulation of the blood.2. The method according to claim 1 wherein the SdrG protein is theligand-binding A region of SdrG.
 3. The method according to claim 1wherein the A region has the sequence of residues 50-597 of SdrG.
 4. Themethod according to claim 1 wherein the SdrG protein is a recombinantprotein.
 5. The method according to claim 1 wherein the method is usedto treat a disease condition selected from the group consisting ofstroke, myocardial infarction, sickle-cell crisis and venous thrombosis.6. The method according to claim 1 wherein the method is used to reducethe concentration of plasma fibrinogen in the patient's blood.
 7. Themethod according to claim 1 wherein the method is used to interfere withor inhibit the release of fibrinopeptide B from fibrinogen.
 8. Themethod according to claim 1 further comprising the step of administeringancrod in an amount effective to interfere or inhibit the release offibrinopeptide A from fibrinogen.
 9. A method of treating or preventingthrombin-induced coagulation comprising administering to a human oranimal patient in need thereof a S. epidermidis fibrinogen-bindingprotein from that can bind to the fibrinogen Bβ chain in an amounteffective to treat or prevent thrombin-induced coagulation.
 10. A methodaccording to claim 9 wherein the fibrinogen-binding protein is selectedfrom the group consisting of SdrG, Fbe, and their respective A regions.11. A method according to claim 9 wherein the fibrinogen-binding proteinbinds at the site of residues 6-20 of the fibrinogen Bβ chain.
 12. Amethod of inhibiting thrombin binding to fibrinogen comprisingadministering to a human or animal patient in need thereof an SdrGprotein in an amount effective to inhibit thrombin binding tofibrinogen.
 13. A method of interfering with or inhibiting the releaseof fibrinopeptide B from fibrinogen comprising administering to a humanor animal patient in need thereof an SdrG protein in an amount effectiveto interfere with or inhibit the release of fibrinopeptide B fromfibrinogen.
 14. A therapeutic composition for treating or preventingthrombin-induced coagulation comprising SdrG in an amount effective totreat or prevent thrombin-induced coagulation, and a pharmaceuticallyacceptable vehicle, carrier or excipient.
 15. A method of treating orpreventing thrombin-induced coagulation comprising administering to ahuman or animal patient in need thereof the composition of claim 14 inan amount effective to treat or prevent thrombin-induced coagulation.16. A method for treating a disease condition selected from the groupconsisting of stroke, myocardial infarction, sickle-cell crisis andvenous thrombosis comprising administering to a human or animal patientin need thereof an effective amount of the composition of claim
 14. 17.A method for reducing plasma fibrinogen concentration in bloodcomprising administering to a human or animal an effective amount of thecomposition according to claim
 14. 18. A therapeutic composition fortreating or preventing thrombin-induced coagulation comprising afibrinogen-binding protein from S. epidermidis which can bind to the Bβchain of fibrinogen in an amount effective to treat or preventthrombin-induced coagulation, and a pharmaceutically acceptable vehicle,carrier or excipient.
 19. The therapeutic composition according to claim18 wherein the fibrinogen-binding protein is selected from the groupconsisting of SdrG, Fbe and their respective A regions.
 20. Thetherapeutic composition according to claim 18 wherein the SdrG A regionhas the sequence of residues 50-597 of SdrG.
 21. The therapeuticcomposition according to claim 18 wherein the fibrinogen-binding proteinis a recombinant protein.
 22. The therapeutic composition according toclaim 18 wherein the fibrinogen-binding protein binds at the site ofresidues 6-20 of the fibrinogen Bβ chain
 23. A method of treating orpreventing thrombin-induced coagulation comprising administering to ahuman or animal patient in need thereof the composition of claim 18 inan amount effective to treat or prevent thrombin-induced coagulation.24. A method for treating a disease condition selected from the groupconsisting of stroke, myocardial infarction, sickle-cell crisis andvenous thrombosis comprising administering to a human or animal patientin need thereof an effective amount of the composition of claim
 18. 25.A method for reducing plasma fibrinogen concentration in bloodcomprising administering to a human or animal an effective amount of thecomposition according to claim 18.